During the metabolism of N-Hydroxy-2-acetylaminofluorene (NOH-2AAF) to the N-O-sulfate ester, a hitherto undiscovered metabolite of NOH-2AAF. Ascorbic acid inhibited formation of the metabolite, while 2-acetylamino-fluorene (2AAF) increased the formation of the metabolite. 3H 2AAF condensed with 14C NOH-2AAF under sulfation conditions to produce the metabolite. It was determined by mass spectrometry and nuclear magnetic resonance spectrometry that the metabolite was 1-(N-2-fluorenylacetamido)-2-fluorenyl-acetamide, a dimer of 2AAF. This dimer was activated to a mutagen by rat liver 9000 x g supernatant in the Ames-Salmonella mutagenesis test system.